We will study the intracellular regulation of platelet function with particular emphasis on the interaction of cytoplasmic ionized calcium, cyclic AMP, and products of arachidonic acid metabolism and other lipids. Direct measurement of the free calcium levels in cytoplasm of platelets and rat promegakaryoblasts (grown in culture) will be carried out using Quin 2 and aequorin. Aequorin will be introduced into the cells by a novel technique requiring permeabilization by EGTA. Parallel experiments involved the two methods for calcium concentration determinaton will permit cross validation of each technique and will allow characterization of the capacities and limitations of the two methods. Studies of platelet calcium homeostasis will be correlated with platelet function including aggregation, secretion, clot retraction and biochemical changes including activation of contractile proteins.